Neurons release neurotransmitter into the synaptic cleft by exocytosis of synaptic vesicles. The proteins that mediate exocytosis are members of conserved protein families involved in general intracellular fusion events. Critical proteins in this process include the SNARE proteins, syntaxin, SNAP-25 and synaptobrevin, as well as UNC-18 and UNC-13. Although we know that UNC-18 and UNC-13 interact with the essential SNARE protein syntaxin, the precise role and sequential order in which these proteins regulate fusion have yet to be determined. In my laboratory we combine genetic and molecular approaches with a newly developed electrophysiological technique to study exocytosis in the nematode Caenorhabditis elegans. Using these techniques we have demonstrated that exocytosis is arrested at a late stage in unc-13 mutants. We now propose to examine the role of UNC-18 in C. elegans synaptic transmission. UNC-18 binds to a closed conformation of syntaxin, which excludes the formation of the SNARE complex. Several models have been proposed for UNC-18 function: (1) UNC-18 dissociation may promote or maintain syntaxin in an open state, enabling SNARE complex assembly and fusion. (2) UNC-18 may maintain syntaxin in the closed conformation to prevent SNARE complex formation. (3) UNC- 18 and syntaxin may mediate the fusion step directly. To test these models, we propose to: 1) Determine whether UNC-18 promotes exocytosis after vesicles have docked to the plasma membrane. 2) Test whether the UNC-18-syntaxin interaction plays an inhibitory role in exocytosis. 3) Test whether constitutively open syntaxin bypasses the requirement for UNC-18. 4) Identify other proteins that act downstream or in parallel with UNC-18 by a) mapping and identifying a recently isolated unc-18 mutant suppressor and b) conducting a genetic screen to identify other unc-18 suppressors. These experiments may contribute to our understanding of Alzheimer's disease, stroke and vesicle trafficking disorders.